Activation & Proliferation of Hematopoietic Stem Cells

描述(申请人提供)：造血干细胞(HSC)与胚胎干细胞不同，具有有限的分裂潜力。在再生障碍性贫血和慢性髓系白血病等血液病中，限制HSC的复制能力似乎很重要。这些限制可能进一步阻碍新的治疗策略的发展，包括体外干细胞扩增和基因治疗。基于这些考虑，可能有助于确定和扩展HSC复制潜力的研究是重要的和普遍感兴趣的。之前由这笔资金资助的纯化的人“候选”HSC的研究表明，HSC的功能特性在个体发育过程中发生了巨大变化，HSC增殖能力随年龄的丧失与可测量的端粒缩短相关。在这里，我们建议进一步研究端粒酶和端粒在造血中的作用。具体地说，我们想测试这一假设，即HSC的复制历史可以通过对其端粒长度的研究来追踪。为了验证这一假设，我们将使用我们实验室开发的精细的流式细胞术技术，检测纯化的HSC及其培养的后代相对于确定的更成熟细胞群体的端粒长度。这些技术还将被用来进一步研究正常人、各种血液疾病患者和基因正常狒狒的家系中有核血细胞端粒长度随年龄的下降。我们将进一步尝试使用基因转移和蛋白质转导策略来操纵HSC的端粒长度，并在体外和体内研究端粒延长的细胞的功能特性。其具体目的是：1)研究正常人和各种血液病患者治疗前后纯化的HSC和有核血细胞的端粒长度；2)研究狒狒有核血细胞的端粒长度与年龄和基因类型的关系；3)研究人工端粒延长和端粒酶抑制在体外和体内对纯化的“候选”HSC增殖、分化和复制能力的影响。这些信息与对造血的基本了解以及干细胞在血液学内外的应用有关。

DESCRIPTION (provided by applicant): Hematopoietic stern cells (HSC), unlike e.g. embryonic stem cells, have a finite potential to divide. Limitations in the replication potential of HSC appear to be important in hematological disorders such as aplastic anemia and chronic myeloid leukemia. Such limitations could furthermore hamper the development of novel therapeutic strategies, including ex vivo stem cell expansion and gene therapy. Based on these considerations, studies that may help define and extend the replicative potential of HSC are important and a general interest. Previous studies with purified human "candidate" HSC funded by this grant have shown that the functional properties of HSC change dramatically during ontogeny and that the loss in HSC proliferative potential with age correlates with measurable shortening of telomeres. Here we propose to further examine the role of telomerase and telomeres in hematopoiesis. Specifically, we want to test the hypothesis that the replication history of HSC can be traced by studies of their telomere length. In order to test this hypothesis, we will examine the telomere length in subsets of purified HSC and their cultured progeny relative to defined populations of more mature cells using refined flow cytometry techniques developed in our laboratory. These techniques will also be used to further study the age related decline in telomere length in nucleated blood cells from normal individuals, patients with various hematological disorders and pedigrees of genotyped normal baboons. We will furthermore attempt to manipulate the telomere length in HSC using gene transfer and protein transduction strategies and study the functional properties of cells with extended telomeres in vitro and in vivo. The specific aims are:1) To study the telomere length in purified HSC and nucleated blood cells from normal individuals and patients with various hematological disorders before and after therapy.2) To study the telomere length in nucleated blood cells from baboons in relation to their age and genotype.3) To study the effect of artificial telomere elongation and telomerase inhibition on the proliferation, differentiation and replicative potential of purified "candidate" HSC in vitro and in vivo.Taken together, these studies will provide crucial baseline information on the role of telomeres in the biology of HSC. Such information is relevant for a basic understanding of hematopoiesis as well as applications of stem cells in and outside hematology.